Our research addresses the cellular and molecular mechanisms that underlie pain hypersensitivity associated with injury and disease. We have been studying the role of the neurotransmitter glutamate (Glu) in the modulation of pain sensitivity in the periphery. Glu is a key inflammatory mediator that is released into peripheral tissues during inflammation, and we have found that G protein-coupled Glu receptors known as metabotropic Glu (mGlu) receptors are expressed in peripheral terminals of nociceptors. In the previous term of this grant, our studies showed that activation of peripheral mGluS in nociceptor terminals induces hypersensitivity to thermal and mechanical stimuli. The thermal hyperalgesia can be explained, at least in part, by PKA-dependent sensitization of the heat-sensing protein, TRPV1. We showed that PKA and PKC directly phosphorylate TRPV1, and identified phosphorylation sites critical for modulation of TRPV1 by PKA and PKC in heterologous systems. However, differences in the PKA and PKC modulation of TRPV1 in heterologous systems relative to natively expressed TRPV1 call into question whether the same phosphorylation sites are involved. While this TRPV1 modulation can account for regulation of thermal nociception by mGluS, it cannot explain the induction of mechanical hypersensitivity. Preliminary data show that mGluS enhances excitability of nociceptors, and we suggest that this may underlie the induction of mechanical sensitization by mGluS. Our work and the work of others points to an important role of mGluS is mediating pain hypersensitivity, and as a consequence mGluS antagonists are being pursued as a novel class of analgesics. However, recent work has called into question whether mGluS antagonists reduce pain by blocking mGluS or by an "off target" action. Studies in the present proposal will address the following open questions: 1) What phosphorylation sites mediate sensitization of TRPV1 in native DRG neurons? 2) What is the relative importance of central and peripheral mGluS in pain hypersensitivity? 3) What are the cellular mechanisms that underlie mechanical hypersensitivity induced by activation of peripheral mGluS? These studies will reveal the cellular and molecular mechanisms by which mGluS modulates thermal and mechanical pain sensation, and will clearly define the role of central and peripheral mGluS in the modulation of pain. They may also help promote the development of mGluS antagonists as analgesics.